Several projects are planned which will evaluate the role of an electrochemical proton gradient across the mitochondrial membrane as an intermediate in energy coupling in oxidative phosphorylation. Kinetic measurements of proton translocation and of ATP synthesis driven by an artificially produced proton gradient will be the most direct test to date of whether this gradient is an intermediate. Energy coupling in reconstituted phospholipid vesicles containing cytochrome oxidase, or NADH-coenzyme Q reductase from the mitochondrial respiratory chain will be further characterized by studies of ion translocation, the effect of ion gradients on redox equilibria and the role of different lipids in the ion transport reaction of these systems. We are also beginning a study of energy conservation in Escherichia coli, and have developed an inverted preparation of the E. coli membrane for studies of oxidative phosphorylation and ion transport.